For the purpose of quick stabilization, biological samples for microscopic, especially electron microscopic, investigations are, to an increasing extent, frozen extremely quickly (shock freezing). Subsequently, the water contained in the samples (often more than 90% by weight) is exchanged at low temperatures for suitable organic organic solvents, for example, water-free acetone or organic solutions, for example osmiumtetroxyde in water-free acetone. This exchange, at least in the critical initial phase, is completed at temperatures considerably under -30.degree. C., since at temperatures over -30.degree., changes in the molecular structure of the objects can occur which reduce the value of the subsequent investigations. Therefore, to be on the safe side, the critical initial phase of the cryo-substitution often takes place in the temperature range between -80.degree. and -120.degree. C., which with the usual cold thermostats cannot be achieved at all, or only with great expenditure of equipment. The proportion in which this expenditure stands in relation to the small measurements and the limited number of such samples, does not justify the employment of expensive and bulky apparatus. Usually, for electron microscopy, for example, from one to ten tissue blocks with an individual volume of between 0.1 and 10 mm.sup.3 are subjected to a cryo-substitution in one operation, which requires, according to the size of the objects and the substitution temperature, between three days and three weeks. During the stated time, the temperature must on no account exceed the respective limiting value to be determined for each object, since otherwise the sample can change in its molecular structure in such a way that reliable scientific statements about its structure in normal life conditions ("in vivo") can no longer be made.
For cryo-substitution up to now, only such laboratory devices have been used, which preferably consist of the samples being placed in closed containers in a freezing mixture of a familiar kind (for example, a dry ice mixture), which has the desired temperature. This procedure requires, however, continuous control as well as at least daily replacement of the freezing mixture. Insofar as the laboratory has at its disposal deep freezers for temperatures under -80.degree., this kind of system is used. Most morphology-oriented laboratories, however, do not have this kind of deep freezer. The acquisition of such a bulky and expensive system is mostly excluded, considering the space requirement, the installation load, as well as the investment costs.
The purpose of the invention under consideration is to make possible a cryo-substitution of small biological samples with an individual volume of under 10 mm.sup.3 for a subsequent, especially electron microscopic investigation, by means of a device which is simple to make and to operate, and which makes possible the requisite low-temperature operation over several days or weeks with the necessary trustworthiness, without routine maintenance and with low routine operating costs.
According to the invention, this purpose is achieved in a device of the kind described above in that the receptacle is filled with a substitution medium, is mounted above the level of the cryogen in the dewar flask, and can be regulated to a temperature between -30.degree. and -120.degree. C. by a cold gas atmosphere circulating around it.
According to the invention there ensues, therefore, an exchange of the ice contained in the frozen biological samples for an organic fluid or solution, the substitution medium, that is poured into the receptacle with the objects. The temperature of between -+.degree. and -120.degree. C. required for the substitution process, in the framework of microscopic, especially electron microscopic, investigations, is thereby achieved in that the metal receptacle with the frozen biological samples is suspended in the substitution medium in the dewar flask containing liquid nitrogen or another suitable cryogen with a boiling point under -30.degree. C. The metal receptacle thereby is not immersed in the cryogen, nor are the larger parts of its surfaces in direct, heat-conducting, metallic contact with the cryogen; rather, it is suspended in the dewar flask, above the level of the liquid cryogen in the dewar flask in such a way that it is not bathed or touched by the cryogen. However, it is mounted in such a way that it is constantly bathed by the continuously evaporating gas of the cryogen or respectively by the cold gas atmosphere, and thereby is cooled to the desired temperature.